Retinal neovascularization (NV) and diabetic macular edema are the major pathological features responsible for vision loss in diabetic retinopathy. Accumulating evidence suggests that chronic inflammation and oxidative stress in the retina play important pathogenic roles in diabetic retinopathy. Wnts are a group of secreted, cysteine-rich glycoproteins which bind to the frizzled (Fz) receptors or to the co-receptor complex consisting of Fz and low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6) and regulate multiple physiological and pathological processes including angiogenesis and inflammation. However, the role of the wnt signaling in diabetic retinopathy has not been reported. Our preliminary studies have provided the following evidence suggesting a pathogenic role of the wnt pathway in diabetic retinopathy: 1) LRP5/6 expression is up-regulated in the retina of streptozotocin (STZ)-induced diabetic rats and of rats with oxygen-induced retinopathy (OIR). 2) A down-stream effector of the wnt pathway, 2-catenin, is accumulated in the retina of STZ-diabetic rats and OIR rats, and is up-regulated by hypoxia in cultured endothelial cells (EC). 3) In cultured EC and in the mouse retina, activation of the wnt pathway alone induces VEGF over-expression. 4) DKK1, a specific inhibitor of the wnt pathway, blocks VEGF expression and decreases retinal NV in the OIR model, and reduces retinal vascular leakage in STZ- diabetic rats. Based on these results, we hypothesize that activation of the wnt pathway plays a pathogenic role in retinal inflammation, vascular leakage and NV in diabetic retinopathy. To test this hypothesis, we propose the following studies: 1) To establish the causative role of the wnt signaling in diabetic retinopathy. We will first identify retinal cell types in which the wnt pathway activation occurs in diabetes. To further establish the role of the wnt pathway in diabetic retinopathy, we will activate the wnt pathway using a constitutively active mutant of 2-catenin and to investigate if activation of the wnt pathway alone causes retinal inflammation, vascular leakage and NV in normal animals. Further, we will block the wnt pathway using DKK1, a specific inhibitor of the wnt pathway, to determine if it attenuates the retinal inflammation and vascular leakage in STZ-diabetic rats and Akita mice, and mitigates retinal NV in OIR rats. 2) To elucidate the molecular mechanism by which the wnt pathway mediates diabetic retinopathy. We will first identify the co-receptor of the wnt, LRP5 or LRP6, essential for induction of diabetic retinopathy. We will knock down the expression of LRP5 and LRP6 using specific siRNAs in cultured EC, to determine which siRNA blocks the VEGF over-expression induced by hypoxia. We will express a constitutively active mutant of LRP5 or LRP6 in the retina of transgenic mice to determine which mutant causes retinal inflammation, vascular leakage and NV. Further, we will cross LRP5-/- mice with Akita mice to determine if LRP5 knockout (KO) attenuates vascular leakage and retinal inflammation induced by diabetes. To investigate the role of LRP5 in retinal NV, newborn LRP5-/- mice will be subjected to OIR to reveal if LRP5 KO prevents or mitigates ischemia-induced retinal NV. Next, we will investigate if the VEGF expression induced by the wnt pathway is through hypoxia-inducible factor-1 (HIF-1). We will knock out HIF-11 in EC cultured from HIF-11 floxed mice, and investigate if the lack of HIF-11 attenuates VEGF over-expression induced by the wnt signaling. We will also investigate if VEGF is essential for mediating the pathogenic role of the wnt signaling in diabetic retinopathy. For this purpose, we will cross Muller cell-specific conditional VEGF-/- mice with the transgenic mice expressing the constitutively active mutant of LRP5 or LRP6 to activate the wnt pathway in VEGF-/- mice and determine if VEGF KO blocks the retinal vascular leakage, inflammation and NV induced by the wnt pathway activation. 3) To elucidate the mechanism by which the wnt pathway is activated in diabetes. We hypothesize that oxidative stress is responsible for the wnt pathway activation in diabetic retinopathy. We will use anti-oxidants in primary EC cultured in high glucose medium or express anti-oxidant enzymes in the retina of the diabetes models to determine if the suppressed ROS generation can inhibit the wnt pathway activation. This project represents an innovative approach to explore the pathogenesis of diabetic retinopathy. These studies will establish the wnt pathway as a novel pathogenic pathway in diabetic retinopathy. The information generated from this project will not only provide new insights into the pathogenesis of diabetic complications, but also reveal a new target for drug intervention of diabetic retinopathy. PUBLIC HEALTH RELEVANCE: Diabetic retinopathy is a common complication of diabetes and a major cause of blindness in working age population. This project aims to explore a novel pathogenic mechanism, i.e, activation of the wnt signal pathway, for diabetic retinopathy and to reveal a new drug target for its treatment.